With increasing effort directed toward purification and/or in vitro expansion of hematopoietic progenitor cells (HPCs) for either fundamental research or clinical applications, there is an associated, increased need for the development of improved, efficient preservation methods for these cells to provide "cell banks." In addition, with the increase in allograft of purified progenitor cells, the possibility of disease transmission from use of contaminated donor material requires that only cells from infectious disease-screened donors be used. The ability to store frozen cells would allow sufficient time for adequate screening to be performed. Therefore, the overall goal of this continuing research is to further develop and optimize an improved system for cryopreservation and storage of human placental/umbilical cord blood (PCB) derived hematopoietic progenitor cells (HPCs), especially as it relates to improving the retention of normal structure and function of frozen-thawed cells to improve clinical outcome (engraftment). In Phase I, a system was developed which provides (1) an efficient, simplified method of bulk-freezing HPC units, and (2) optimized removal of cryoprotectant (DMSO) while maintaining a closed system. In Phase II, this system will be refined to allow (1) ease of use in the clinical environment, and (2) ease of manufacture/commercialization. It is the goal of this Phase II proposal to refine the closed cryopreservation, storage, and PCB washing system based on sound biophysical cell-type specific characteristics and mathematical modeling which will allow efficient and safe use of cryopreserved PCB derived HPCs. To achieve this goal, the following specific aims are proposed: (1) Validation and refinement of the cryopreservation method/system-using colony forming assays in semi-solid media to determine clonogenic ability, (2) Validation and refinement using a human long-term culture initiating cell (LTC-IC) assay, and (3) Testing of the cryopreservation system by freezing/thawing/washing HPCs and transplanting into a NOD/SCID mouse model.